Histological and immunohistochemical characterization of the porcine ocular surface

Abstract

The ocular surface of the white domestic pig (Sus scrofa domestica) is used as a helpful model of the human ocular surface; however, a complete histological description has yet to be published. In this work, we studied porcine eyeballs with intact eyelids to describe and characterize the different structures that form the ocular surface, including the cornea and conjunctiva that covers the bulbar sclera, tarsi, and the nictitating membrane. We determined the distribution of goblet cells of different types over the conjunctiva and analyzed the conjunctival-associated lymphoid tissue (CALT). Porcine eyeballs were obtained from a local slaughterhouse, fixed, processed, and embedded in paraffin blocks. Tissue sections (4 μm) were stained with hematoxylin/eosin, Alcian blue/Periodic Acid Schiff, and Giemsa. Slides were also stained with lectins from Arachis hypogaea (PNA) and Helix pomatia (HPA) agglutinins and immunostained with rabbit anti-CD3. We found that the porcine cornea was composed of 6-8 epithelial cell layers, stroma, Descemet's membrane, and an endothelial monolayer. The total corneal thickness was 1131.0±87.5 μm (mean±standard error of the mean) in the center and increased to 1496.9±138.2 μm at the limbus. The goblet cell density was 71.25±12.29 cells/mm, ranging from the highest density (113.04±37.21 cells/mm) in the lower palpebral conjunctiva to the lowest density (12.69±4.29 cells/mm) in the bulbar conjunctiva. The CALT was distributed in the form of intraepithelial lymphocytes and subepithelial diffuse lymphoid tissue. Lenticular-shaped lymphoid follicles, about 8 per histological section, were also present within the conjunctival areas. In conclusion, we demonstrated that the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

Fig 1. Macroscopic photograph of porcine eye.

Transverse section of a fixed pig eyeball with the eyelids. The white line marks the limits of the conjunctiva.

Fig 2. Histological analysis of the porcine cornea.

Tissue section stained with hematoxylin-eosin showing the four layers of the cornea.

Fig 3. Histological analysis of the porcine limbus.

Tissue section of porcine limbus stained with Alcian blue/periodic acid Schiff showing the characteristic palisades of Vogt (arrows) where limbal epithelial stem cells reside.

Fig 4. Histological analysis of the porcine ocular surface.

Low magnification tissue section of porcine anterior ocular surface stained with Alcian blue/periodic acid Schiff. Bar = 1 mm.

Fig 5. Histological analysis of porcine conjunctiva.

Tissue sections of pig conjunctiva stained with AB/PAS. (A) The marginal conjunctiva between the tarsal and palpebral surfaces was covered by a stratified squamous epithelium and deeper cuboidal epithelial cells. Epithelial downgrowths into the stroma appeared as crypts (arrowheads). (B) The tarsal conjunctiva had a large number of goblet cells containing acidic glycoconjugates. (C) Conjunctiva in the fornix. (D) The bulbar conjunctiva had 4 epithelial cell layers and very few goblet cells.

Fig 6. Porcine nictitating membrane.

(A) Low magnification micrograph of the nictitating membrane union with upper eyelid. Bar = 500 μm. (B) Goblet cells in the conjunctival epithelium over the nictitating membrane cartilage. (C) Conjunctival edge between nictitating membrane and lower eyelid. (D) Conjunctiva over the center of the nictitating membrane anterior surface showing great abundance of goblet cells containing acidic, neutral, or both types of glycoconjugates.

Fig 7. Porcine conjunctival goblet cells.

(A) The different types of goblet cells can be distinguished with AB/PAS staining. Acidic glycoconjugates were stained blue (arrow) by AB, and neutral glycoconjugates were pink (arrowhead) by PAS. Most goblet cells have both types of glycoconjugate granules and appear as dark blue or purple color. (B) H/E staining showed a pseudogland of Henle (arrows) formed by a group of goblet cells embedded within the conjunctival stroma.

Fig 8. Goblet cell density (GCD) in the different conjunctival regions.

The bar at 71.25 cells/mm represents the mean GCD for the whole conjunctiva. p ≤ 0.005 for *, vs upper palpebral; &, vs lower palpebral; #, vs bulbar.

Fig 9. Lectin binding in porcine conjunctiva.

Lectins from Arachis hypogaea agglutinin (PNA, green) and Helix pomatia agglutinin (HPA, red) bound to porcine lower palpebral conjunctival goblet cells.

Fig 10. CALT in porcine conjunctiva.

(A) The palpebral conjunctiva contained diffuse lymphoid tissue (asterisks) and a follicle. (B) Conjunctival section stained with H/E, showing the presence of CALT in the porcine conjunctiva. A diffuse lymphoid layer was present underneath the epithelium. In this representative image, there was a well-developed follicle in the stroma, and the characteristics of the follicle-associated epithelium (FAE) were evident. FAE, follicle-associated epithelium. (C) Tissue section of palpebral conjunctiva stained with Giemsa showing the presence of a well-defined follicle and HEVs (arrows).

Fig 11. CD3 immunofluorescence in porcine conjunctiva.

Dotted line marks the limits of the same follicle shown in each of the panels. Dotted-dash lines mark the limit between conjunctival epithelium and stroma. (A) nuclei [blue], (B) Negative control of CD3⁺ lymphocytes [green], and (C) merged images of (A) and (B). (D) nuclei [blue], (E) CD3⁺ lymphocytes [green], and (F) merged images of (D) and E). (G) nuclei [blue], (H) CD3⁺ lymphocytes [green], and (I) merged images of (G) and (H).

Fig 12. Meibomian glands in the pig eye.

Different meibocyte acini (arrows) surround the Meibomian gland ductules (asterisks). Areas of meibocyte disintegration were clearly evident (pluses).